In this application we propose to extend our studies of tubulin self-assembly from the realm of nucleotide hydrolysis and the polymer elongations reactions with the goal of considereing two less characterized phases of the self-assembly process. These are: the nucleation (or early initiation) phase and the dynamics of steady-state turnover of assembled microtubules. Our specific aims include the extension of our singlet-singlet fluorescence energy transfer experiments with dye labeled tubulin protomers, ring-structures, and assembled microtubules. We also plan complementary radiochemical and turbidity studies of the kinetic properties of the so-called ring (oligomeric forms of tubulin with as yet undefined roles in the self-assembly process) to uncover more of the information expressed in the early course of microtubule formation. We also planto derive new information about the nature of the protomer-to-polymerization nucleus transition using methods to maintain the free unpolymerized tubulin protomer concentration at varius predetermined levels rather than permitting the tubulin concentration to change during assembly. We also plan to carefully examine for the occurrence of tru nucleation phenomena (in the physico-chemical sense) by studying protomer-polymer coexistence curves (i.e., plots of polymer weight concentratio versus protomer concentration) in both the assembly and dilution-induced disassembly directions. With respect to the dynamic properties of assembled microtubules, we propose to extend our earlier work with GTP and nonhydrolyzable guanine nucleotide analogues and to further extend our studies of rapid microtubule disassembly. Our plans also include a detailed characterization of solution variable effects on the steady-state head-to-tail polymerization process (sometimes referred to as opposite-end assembly/disassembly or more loosely as "treadmilling"). The development of appropriate rate theory and the derivation of new rate laws for polymer-protomer interactions will remain of continuing interest throughout these efforts.